Here we present a method of modeling fluorophore molecules as spheres in evan escent fields mathematically instead of the assumption of tophat function dist r ibution. Based on this model, we discuss that it is possible to calculate relati ve separation distances of the molecules, and their axial positions and diameters with the total internal reflection fluorescence microscopy(TIRFM) due to its uni que intensity pattern in the z direction. In the whole process, we need mere ly to change the incident angle once, that is to get only two frame imaging of TIRFM.